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1. Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

   https://doi.org/10.1093/nar/gkab041  

   Ketkar, Amit; Smith, Lane; Johnson, Callie; Richey, Alyssa; Berry, Makayla; Hartman, Jessica H; Maddukuri, Leena; Reed, Megan R; Gunderson, Julie E; Leung, Justin W; et al (February 2021, Nucleic Acids Research)

   null (Ed.)

   Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication. 

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2. Role of Resultant Dipole Moment in Mechanical Dissociation of Biological Complexes

   https://doi.org/10.3390/molecules23081995  

   Kouza, Maksim; Banerji, Anirban; Kolinski, Andrzej; Buhimschi, Irina; Kloczkowski, Andrzej (August 2018, Molecules)

   Protein-peptide interactions play essential roles in many cellular processes and their structural characterization is the major focus of current experimental and theoretical research. Two decades ago, it was proposed to employ the steered molecular dynamics (SMD) to assess the strength of protein-peptide interactions. The idea behind using SMD simulations is that the mechanical stability can be used as a promising and an efficient alternative to computationally highly demanding estimation of binding affinity. However, mechanical stability defined as a peak in force-extension profile depends on the choice of the pulling direction. Here we propose an uncommon choice of the pulling direction along resultant dipole moment (RDM) vector, which has not been explored in SMD simulations so far. Using explicit solvent all-atom MD simulations, we apply SMD technique to probe mechanical resistance of ligand-receptor system pulled along two different vectors. A novel pulling direction—when ligand unbinds along the RDM vector—results in stronger forces compared to commonly used ligand unbinding along center of masses vector. Our observation that RDM is one of the factors influencing the mechanical stability of protein-peptide complex can be used to improve the ranking of binding affinities by using mechanical stability as an effective scoring function. 

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3. Structural insights into binding of polyglutamylated tetrahydrofolate by serine hydroxymethyltransferase 8 from soybean

   https://doi.org/10.3389/fpls.2024.1451839  

   Owuocha, Luckio F; Mitchum, Melissa G; Beamer, Lesa J (August 2024, Frontiers in Plant Science)

   Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate. Here, we use X-ray crystallography to provide a high-resolution view of interactions between the enzyme serine hydroxymethyltransferase (SHMT), which provides one carbon precursors for the folate cycle, and a polyglutamylated form of THF. Our 1.7 Å crystal structure of soybean SHMT8 in complex with diglutamylated 5-formyl-THF reveals, for the first time, a structural rearrangement of a loop at the entrance to the folate binding site accompanied by the formation of novel specific interactions between the enzyme and the diglutamyl tail of the ligand. Biochemical assays show that additional glutamate moieties on the folate ligand increase both enzyme stability and binding affinity. Together these studies provide new information on SHMT structure and function and inform the design of anti-folate agents. 

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4. Protein‐Ligand Structure and Affinity Prediction in CASP16 Using a Geometric Deep Learning Ensemble and Flow Matching

   https://doi.org/10.1002/prot.26827  

   Morehead, Alex; Liu, Jian; Neupane, Pawan; Giri, Nabin; Cheng, Jianlin (April 2025, Proteins: Structure, Function, and Bioinformatics)

   ABSTRACT Predicting the structure of ligands bound to proteins is a foundational problem in modern biotechnology and drug discovery, yet little is known about how to combine the predictions of protein‐ligand structure (poses) produced by the latest deep learning methods to identify the best poses and how to accurately estimate the binding affinity between a protein target and a list of ligand candidates. Further, a blind benchmarking and assessment of protein‐ligand structure and binding affinity prediction is necessary to ensure it generalizes well to new settings. Towards this end, we introduceMULTICOM_ligand, a deep learning‐based protein‐ligand structure and binding affinity prediction ensemble featuring structural consensus ranking for unsupervised pose ranking and a new deep generative flow matching model for joint structure and binding affinity prediction. Notably,MULTICOM_ligand ranked among the top‐5 ligand prediction methods in both protein‐ligand structure prediction and binding affinity prediction in the 16th Critical Assessment of Techniques for Structure Prediction (CASP16), demonstrating its efficacy and utility for real‐world drug discovery efforts. The source code for MULTICOM_ligand is freely available on GitHub. 

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5. “Turbo‐Charged” DNA Motors with Optimized Sequence Enable Single‐Molecule Nucleic Acid Sensing

   https://doi.org/10.1002/anie.202316851  

   Zhang, Luona; Piranej, Selma; Namazi, Arshiya; Narum, Steven; Salaita, Khalid (March 2024, Angewandte Chemie International Edition)

   Abstract DNA motors that consume chemical energy to generate processive mechanical motion mimic natural motor proteins and have garnered interest due to their potential applications in dynamic nanotechnology, biosensing, and drug delivery. Such motors translocate by a catalytic cycle of binding, cleavage, and rebinding between DNA “legs” on the motor body and RNA “footholds” on a track. Herein, we address the well‐documented trade‐off between motor speed and processivity and investigate how these parameters are controlled by the affinity between DNA legs and their complementary footholds. Specifically, we explore the role of DNA leg length and GC content in tuning motor performance by dictating the rate of leg‐foothold dissociation. Our investigations reveal that motors with 0 % GC content exhibit increased instantaneous velocities of up to 150 nm/sec, three‐fold greater than previously reported DNA motors and comparable to the speeds of biological motor proteins. We also demonstrate that the faster speed and weaker forces generated by 0 % GC motors can be leveraged for enhanced capabilities in sensing. We observe single‐molecule sensitivity when programming the motors to stall in response to the binding of nucleic acid targets. These findings offer insights for the design of high‐performance DNA motors with promising real‐world biosensing applications. 

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